Novel human proteases and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/244,939 which was filed on Nov. 1, 2000 and is hereinincorporated by reference in its entirety.

INTRODUCTION

[0002] The present invention relates to the discovery, identification,and characterization of novel human polynucleotides encoding proteinssharing sequence similarity with mammalian meltrin-beta/ADAM 19homologue metalloproteases. The invention encompasses the describedpolynucleotides, host cell expression systems, the encoded proteins,fusion proteins, polypeptides and peptides, antibodies to the encodedproteins and peptides, and genetically engineered animals that eitherlack or over express the disclosed sequences, antagonists and agonistsof the proteins, and other compounds that modulate the expression oractivity of the proteins encoded by the disclosed polynucleotides thatcan be used for diagnosis, drug screening, clinical trial monitoring,the treatment of diseases and disorders, and cosmetic or nutriceuticalapplications.

BACKGROUND OF THE INVENTION

[0003] Proteases cleave protein substrates as part of degradation,maturation, and secretory pathways within the body. Proteases have beenassociated with, inter alia, regulating development, diabetes, obesity,infertility, modulating cellular processes, and infectious disease.

SUMMARY OF THE INVENTION

[0004] The present invention relates to the discovery, identification,and characterization of nucleotides that encode novel human proteins,and the corresponding amino acid sequences of these proteins. The novelhuman proteins (NHPS) described for the first time herein sharestructural similarity with animal proteases and particularlymetalloproteases and disintegrins such as meltrin-beta and ADAM 19.

[0005] The novel human nucleic acid (cDNA) sequences described herein,encode proteins/open reading frames (ORFS) of 926, 918, 963, and 955amino acids in length (see SEQ ID NOS: 2, 4, 6, and 8 respectively).

[0006] The invention also encompasses agonists and antagonists of thedescribed NHPs, including small molecules, large molecules, mutant NHPs,or portions thereof, that compete with native NHP, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHPs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stemcells (“ES cells”) lines that contain gene trap mutations in a murinehomolog of at least one of the described NHPs. When the unique NHPsequences described in SEQ ID NOS:1-9 are “knocked-out” they provide amethod of identifying phenotypic expression of the particular gene aswell as a method of assigning function to previously unknown genes. Inaddition, animals in which the unique NHP sequences described in SEQ IDNOS:1-9 are “knocked-out” provide a unique source in which to elicitantibodies to homologous and orthologous proteins which would have beenpreviously viewed by the immune system as “self” and therefore wouldhave failed to elicit significant antibody responses. To these ends,gene trapped knockout ES cells have been generated in murine homologs ofthe described NHPS.

[0007] Additionally, the unique NHP sequences described in SEQ IDNOS:1-9 are useful for the identification of protein coding sequence andmapping a unique gene to a particular chromosome (the gene encoding thedescribed NHPs is apparently encoded on human chromosome 5, see GENBANKaccession no. AC008676). These sequences identify actual, biologicallyrelevant, exon splice junctions as opposed to those that might have beenpredicted bioinformatically from genomic sequence alone. The sequencesof the present invention are also useful as additional DNA markers forrestriction fragment length polymorphism (RFLP) analysis, and inforensic biology.

[0008] Further, the present invention also relates to processes foridentifying compounds that modulate, i.e., act as agonists orantagonists, of NHP expression and/or NHP activity that utilize purifiedpreparations of the described NHPs and/or NHP product, or cellsexpressing the same. Such compounds can be used as therapeutic agentsfor the treatment of any of a wide variety of symptoms associated withbiological disorders or imbalances.

DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0009] The Sequence Listing provides the sequences of several NHP ORFsencoding the described NHP amino acid sequences. SEQ ID NO:9 describes aNHP ORF and flanking sequences.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The NHP sequences described for the first time herein are novelproteins that are expressed in, inter alia, human cell lines, and humanfetal brain, brain, pituitary, cerebellum, spinal cord, thymus, spleen,lymph node, bone marrow, trachea, lung, kidney, fetal liver, liver,prostate, testis, adrenal gland, pancreas, salivary gland, stomach,small intestine, colon, skeletal muscle, heart, uterus, placenta,mammary gland, skin, adipose, esophagus, bladder, cervix, rectum,hypothalamus, ovary, fetal kidney, gall bladder, tongue, carcinomacells, umbilical vein, endothelium, and fetal lung cells.

[0011] The described sequences were compiled from cDNA clones, genomicsequence, and cDNAs derived from human fetal brain, testis, mammarygland, placenta, adipose, uterus, skeletal muscle, fetus, kidney, brain,thymus, and adrenal gland mRNAs (Edge Biosystems, Gaithersburg, Md., andClontech, Palo Alto, Calif.). The present invention encompasses thenucleotides presented in the Sequence Listing, host cells expressingsuch nucleotides, the expression products of such nucleotides, and: (a)nucleotides that encode mammalian homologs of the described genes,including the specifically described NHPs, and NHP products; (b)nucleotides that encode one or more portions of a NHP that correspond tofunctional domains, and the polypeptide products specified by suchnucleotide sequences, including but not limited to the novel regions ofany active domain(s); (c) isolated nucleotides that encode mutantversions, engineered or naturally occurring, of the described NHPs inwhich all or a part of at least one domain is deleted or altered, andthe polypeptide products specified by such nucleotide sequences,including but not limited to soluble proteins and peptides in which allor a portion of the signal sequence is deleted; (d) nucleotides thatencode chimeric fusion proteins containing all or a portion of a codingregion of a NHP, or one of its domains (e.g., a receptor or ligandbinding domain, accessory protein/self-association domain, etc.) fusedto another peptide or polypeptide; or (e) therapeutic or diagnosticderivatives of the described polynucleotides such as oligonucleotides,antisense polynucleotides, ribozymes, dsRNA, or gene therapy constructscomprising a sequence first disclosed in the Sequence Listing.

[0012] As discussed above, the present invention includes: (a) the humanDNA sequences presented in the Sequence Listing (and vectors comprisingthe same) and additionally contemplates any nucleotide sequence encodinga contiguous NHP open reading frame (ORF), or a contiguous exon splicejunction first described in the Sequence Listing, that hybridizes to acomplement of a DNA sequence presented in the Sequence Listing underhighly stringent conditions, e.g., hybridization to filter-bound DNA in0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3)and encodes a functionally equivalent expression product. Additionallycontemplated are any nucleotide sequences that hybridize to thecomplement of the DNA sequence that encode and express an amino acidsequence presented in the Sequence Listing under moderately stringentconditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al.,1989, supra), yet still encode a functionally equivalent NHP product.Functional equivalents of a NHP include naturally occurring NHPs presentin other species and mutant NHPs whether naturally occurring orengineered (by site directed mutagenesis, gene shuffling, directedevolution as described in, for example, U.S. Pat. No. 5,837,458). Theinvention also includes degenerate nucleic acid variants of thedisclosed NHP polynucleotide sequences.

[0013] Additionally contemplated are polynucleotides encoding a NHP ORF,or its functional equivalent, encoded by a polynucleotide sequence thatis about 99, 95, 90, or about 85 percent similar or identical tocorresponding regions of the nucleotide sequences of the SequenceListing (as measured by BLAST sequence comparison analysis using, forexample, the GCG sequence analysis package using standard defaultsettings).

[0014] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the described NHP gene nucleotide sequences. Such hybridizationconditions may be highly stringent or less highly stringent, asdescribed above. In instances where the nucleic acid molecules aredeoxyoligonucleotides (“DNA oligos”), such molecules are generally about16 to about 100 bases long, or about 20 to about 80, or about 34 toabout 45 bases long, or any variation or combination of sizesrepresented therein that incorporate a contiguous region of sequencefirst disclosed in the Sequence Listing. Such oligonucleotides can beused in conjunction with the polymerase chain reaction (PCR) to screenlibraries, isolate clones, and prepare cloning and sequencing templates,etc.

[0015] Alternatively, such NHP oligonucleotides can be used ashybridization probes for screening libraries, and assessing geneexpression patterns (particularly using a micro array or high-throughput“chip” format). Additionally, a series of the described NHPoligonucleotide sequences, or the complements thereof, can be used torepresent all or a portion of the described NHP sequences. Anoligonucleotide or polynucleotide sequence first disclosed in at least aportion of one or more of the sequences of SEQ ID NOS: 1-9 can be usedas a hybridization probe in conjunction with a solid supportmatrix/substrate (resins, beads, membranes, plastics, polymers, metal ormetallized substrates, crystalline or polycrystalline substrates, etc.).Of particular note are spatially addressable arrays (i.e., gene chips,microtiter plates, etc.) of oligonucleotides and polynucleotides, orcorresponding oligopeptides and polypeptides, wherein at least one ofthe biopolymers present on the spatially addressable array comprises anoligonucleotide or polynucleotide sequence first disclosed in at leastone of the sequences of SEQ ID NOS: 1-9, or an amino acid sequenceencoded thereby. Methods for attaching biopolymers to, or synthesizingbiopolymers on, solid support matrices, and conducting binding studiesthereon are disclosed in, inter alia, U.S. Pat. Nos. 5,700,637,5,556,752, 5,744,305, 4,631,211, 5,445,934, 5,252,743, 4,713,326,5,424,186, and 4,689,405 the disclosures of which are hereinincorporated by reference in their entirety.

[0016] Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-9 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is within a range of between about 8 to about2000 nucleotides. Preferably the probes consist of 60 nucleotides andmore preferably 25 nucleotides from the sequences first disclosed in SEQID NOS:1-9.

[0017] For example, a series of the described oligonucleotide sequences,or the complements thereof, can be used in chip format to represent allor a portion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length can partially overlap each other and/or thesequence may be represented using oligonucleotides that do not overlap.Accordingly, the described polynucleotide sequences shall typicallycomprise at least about two or three distinct oligonucleotide sequencesof at least about 8 nucleotides in length that are each first disclosedin the described Sequence Listing. Such oligonucleotide sequences canbegin at any nucleotide present within a sequence in the SequenceListing and proceed in either a sense (5′-to-3′) orientation vis-a-visthe described sequence or in an antisense orientation.

[0018] Microarray-based analysis allows the discovery of broad patternsof genetic activity, providing new understanding of gene functions andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-9 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel componentsor gene functions that manifest themselves as novel phenotypes.

[0019] Probes consisting of sequences first disclosed in SEQ ID NOS:1-9can also be used in the identification, selection and validation ofnovel molecular targets for drug discovery. The use of these uniquesequences permits the direct confirmation of drug targets andrecognition of drug dependent changes in gene expression that aremodulated through pathways distinct from the drugs intended target.These unique sequences therefore also have utility in defining andmonitoring both drug action and toxicity.

[0020] As an example of utility, the sequences first disclosed in SEQ IDNOS:1-9 can be utilized in microarrays or other assay formats, to screencollections of genetic material from patients who have a particularmedical condition. These investigations can also be carried out usingthe sequences first disclosed in SEQ ID NOS:1-9 in silico and bycomparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

[0021] Thus the sequences first disclosed in SEQ ID NOS:1-9 can be usedto identify mutations associated with a particular disease and also as adiagnostic or prognostic assay.

[0022] Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence in conjunctionwith the presence of one or more specific oligonucleotide sequence(s)first disclosed in the SEQ ID NOS: 1-9. Alternatively, a restriction mapspecifying the relative positions of restriction endonuclease digestionsites, or various palindromic or other specific oligonucleotidesequences can be used to structurally describe a given sequence. Suchrestriction maps, which are typically generated by widely availablecomputer programs (e.g., the University of Wisconsin GCG sequenceanalysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor, Mich.,etc.), can optionally be used in conjunction with one or more discretenucleotide sequence(s) present in the sequence that can be described bythe relative position of the sequence relative to one or more additionalsequence(s) or one or more restriction sites present in the disclosedsequence.

[0023] For oligonucleotide probes, highly stringent conditions mayrefer, e.g., to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos). These nucleic acid moleculesmay encode or act as NHP gene antisense molecules, useful, for example,in NHP gene regulation (for and/or as antisense primers in amplificationreactions of NHP gene nucleic acid sequences). With respect to NHP generegulation, such techniques can be used to regulate biologicalfunctions. Further, such sequences may be used as part of ribozymeand/or triple helix sequences that are also useful for NHP generegulation.

[0024] Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety which isselected from the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0025] The antisense oligonucleotide can also comprise at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0026] In yet another embodiment, the antisense oligonucleotide willcomprise at least one modified phosphate backbone selected from thegroup consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

[0027] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

[0028] Oligonucleotides of the invention can be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides can be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0029] Low stringency conditions are well known to those of skill in theart, and will vary predictably depending on the specific organisms fromwhich the library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y.

[0030] Alternatively, suitably labeled NHP nucleotide probes can be usedto screen a human genomic library using appropriately stringentconditions or by PCR. The identification and characterization of humangenomic clones is helpful for identifying polymorphisms (including, butnot limited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

[0031] For example, the present sequences can be used in restrictionfragment length polymorphism (RFLP) analysis to identify specificindividuals. In this technique, an individual's genomic DNA is digestedwith one or more restriction enzymes, and probed on a Southern blot toyield unique bands for identification (as generally described in U.S.Pat. No. 5,272,057, incorporated herein by reference). In addition, thesequences of the present invention can be used to provide polynucleotidereagents, e.g., PCR primers, targeted to specific loci in the humangenome, which can enhance the reliability of DNA-based forensicidentifications by, for example, providing another “identificationmarker” (i.e., another DNA sequence that is unique to a particularindividual). Actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments

[0032] Further, a NHP homolog can be isolated from nucleic acid from anorganism of interest by performing PCR using two degenerate or “wobble”oligonucleotide primer pools designed on the basis of amino acidsequences within the NHP products disclosed herein. The template for thereaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of mRNA prepared from human or non-human cell lines ortissue known or suspected to express an allele of a NHP gene. The PCRproduct can be subcloned and sequenced to ensure that the amplifiedsequences represent the sequence of the desired NHP gene. The PCRfragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

[0033] PCR technology can also be used to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express a NHP gene, such as, for example, testistissue). A reverse transcription (RT) reaction can be performed on theRNA using an oligonucleotide primer specific for the most 5′ end of theamplified fragment for the priming of first strand synthesis. Theresulting RNA/DNA hybrid may then be “tailed” using a standard terminaltransferase reaction, the hybrid may be digested with RNase H, andsecond strand synthesis may then be primed with a complementary primer.Thus, cDNA sequences upstream of the amplified fragment can be isolated.For a review of cloning strategies that can be used, see e.g., Sambrooket al., 1989, supra.

[0034] A cDNA encoding a mutant NHP sequence can be isolated, forexample, by using PCR. In this case, the first cDNA strand may besynthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolatedfrom tissue known or suspected to be expressed in an individualputatively carrying a mutant NHP allele, and by extending the new strandwith reverse transcriptase. The second strand of the cDNA is thensynthesized using an oligonucleotide that hybridizes specifically to the5′ end of the normal sequence. Using these two primers, the product isthen amplified via PCR, optionally cloned into a suitable vector, andsubjected to DNA sequence analysis through methods well known to thoseof skill in the art. By comparing the DNA sequence of the mutant NHPallele to that of a corresponding normal NHP allele, the mutation(s)responsible for the loss or alteration of function of the mutant NHPgene product can be ascertained.

[0035] Alternatively, a genomic library can be constructed using DNAobtained from an individual suspected of or known to carry a mutant NHPallele (e.g., a person manifesting a NHP-associated phenotype such as,for example, obesity, high blood pressure, arthritis, asthma, connectivetissue disorders, infertility, etc.), or a cDNA library can beconstructed using RNA from a tissue known, or suspected, to express amutant NHP allele. A normal NHP gene, or any suitable fragment thereof,can then be labeled and used as a probe to identify the correspondingmutant NHP allele in such libraries. Clones containing mutant NHPsequences can then be purified and subjected to sequence analysisaccording to methods well known to those skilled in the art.

[0036] Additionally, an expression library can be constructed utilizingcDNA synthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant NHP allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue can be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against normal NHP product, as described below. (For screeningtechniques, see, for example, Harlow, E. and Lane, eds., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, Cold SpringHarbor.)

[0037] Additionally, screening can be accomplished by screening withlabeled NHP fusion proteins, such as, for example, alkalinephosphatase-NHP or NHP-alkaline phosphatase fusion proteins. In caseswhere a NHP mutation results in an expression product with alteredfunction (e.g., as a result of a missense or a frameshift mutation),polyclonal antibodies to NHP are likely to cross-react with acorresponding mutant NHP expression product. Library clones detected viatheir reaction with such labeled antibodies can be purified andsubjected to sequence analysis according to methods well known in theart.

[0038] The invention also encompasses (a) DNA vectors that contain anyof the foregoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example, baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporated byreference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation) or geneticallyengineered transcription factor. As used herein, regulatory elementsinclude, but are not limited to, inducible and non-inducible promoters,enhancers, operators and other elements known to those skilled in theart that drive and regulate expression. Such regulatory elements includebut are not limited to the cytomegalovirus (hCMV) immediate early gene,regulatable, viral elements (particularly retroviral LTR promoters), theearly or late promoters of SV40 adenovirus, the lac system, the trpsystem, the TAC system, the TRC system, the major operator and promoterregions of phage lambda, the control regions of fd coat protein, thepromoter for 3-phosphoglycerate kinase (PGK), the promoters of acidphosphatase, and the promoters of the yeast α-mating factors.

[0039] The present invention also encompasses antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists andagonists of a NHP, as well as compounds or nucleotide constructs thatinhibit expression of a NHP sequence (transcription factor inhibitors,antisense and ribozyme molecules, or open reading frame sequence orregulatory sequence replacement constructs), or promote the expressionof a NHP (e.g., expression constructs in which NHP coding sequences areoperatively associated with expression control elements such aspromoters, promoter/enhancers, etc.).

[0040] The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotidesequences, antibodies, antagonists and agonists can be useful for thedetection of mutant NHPs or inappropriately expressed NHPs for thediagnosis of disease. The NHPs or NHP peptides, NHP fusion proteins, NHPnucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of NHP inthe body. The use of engineered host cells and/or animals may offer anadvantage in that such systems allow not only for the identification ofcompounds that bind to the endogenous receptor for a NHP, but can alsoidentify compounds that trigger NHP-mediated activities or pathways.

[0041] Finally, the NHP products can be used as therapeutics. Forexample, soluble derivatives such as NHP peptides/domains correspondingto NHP, NHP fusion protein products (especially NHP-Ig fusion proteins,i.e., fusions of a NHP, or a domain of a NHP, to an IgFc), NHPantibodies and anti-idiotypic antibodies (including Fab fragments),antagonists or agonists (including compounds that modulate or act ondownstream targets in a NHP- mediated pathway) can be used to directlytreat diseases or disorders. For instance, the administration of aneffective amount of soluble NHP, or a NHP-IgFc fusion protein or ananti-idiotypic antibody (or its Fab) that mimics a NHP could activate oreffectively antagonize the endogenous NHP receptor. Nucleotideconstructs encoding such NHP products can be used to geneticallyengineer host cells to express such products in vivo; these geneticallyengineered cells function as “bioreactors” in the body delivering acontinuous supply of a NHP, a NHP peptide, or a NHP fusion protein tothe body. Nucleotide constructs encoding functional NHP, mutant NHPs, aswell as antisense and ribozyme molecules can also be used in “genetherapy” approaches for the modulation of NHP expression. Thus, theinvention also encompasses pharmaceutical formulations and methods fortreating biological disorders.

[0042] Various aspects of the invention are described in greater detailin the subsections below.

The Nhp Sequences

[0043] The cDNA sequences and corresponding deduced amino acid sequencesof the described NHPs are presented in the Sequence Listing. The NHPnucleotides were obtained from human cDNA libraries using probes and/orprimers generated from human genomic sequence. Expression analysis hasprovided evidence that the described NHP can be expressed a variety ofhuman cells.

[0044] Several polymorphisms were identified including an A/Gpolymorphism at the nucleotide position represented by, for example,position 313 of SEQ ID NO: 1 (which can result in a thr or ala at theregion corresponding to amino acid (aa) position 105 of, for example,SEQ ID NO:2), an A/T at nucleotide position 1670 (which can result in anasp or val at aa position 557), an A/G at nucleotide position 1864(which can result in an asp or asn at aa position 622), and a T/C atnucleotide position 1915 (which can result in an phe or leu at aaposition 639).

[0045] An additional application of the described novel humanpolynucleotide sequences is their use in the molecularmutagenesis/evolution of proteins that are at least partially encoded bythe described novel sequences using, for example, polynucleotideshuffling or related methodologies. Such approaches are described inU.S. Pat. Nos. 5,830,721 and 5,837,458 which are herein incorporated byreference in their entirety.

[0046] NHP gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, worms, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, birds, goats, andnon-human primates, e.g., baboons, monkeys, and chimpanzees may be usedto generate NHP transgenic animals.

[0047] Any technique known in the art may be used to introduce a NHPtransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

[0048] The present invention provides for transgenic animals that carrythe NHP transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. The transgene may be integrated as asingle transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell type ofinterest, and will be apparent to those of skill in the art.

[0049] When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous NHP gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals).

[0050] The transgene can also be selectively introduced into aparticular cell type, thus inactivating the endogenous NHP gene in onlythat cell type, by following, for example, the teaching of Gu et al.,1994, Science, 265:103-106. The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art.

[0051] Once transgenic animals have been generated, the expression ofthe recombinant NHP gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of NHP gene-expressing tissue, may also beevaluated immunocytochemically using antibodies specific for the NHPtransgene product.

Nhps and Nhp Polypeptides

[0052] The NHPs, NHP polypeptides, NHP peptide fragments, mutated,truncated, or deleted forms of NHP, and/or NHP fusion proteins can beprepared for a variety of uses. These uses include but are not limitedto the generation of antibodies, as reagents in diagnostic assays, theidentification of other cellular gene products related to a NHP, asreagents in assays for screening for compounds that can be used aspharmaceutical reagents useful in the therapeutic treatment of mental,biological, or medical disorders and disease. The described NHPs sharesimilarity with a variety of proteases, including, but not limited to,disintegrins, ADAMs, and fertilins.

[0053] The Sequence Listing discloses the amino acid sequences encodedby the described NHP polynucleotides. The NHPs display initiatormethionines in DNA sequence contexts consistent with translationinitiation sites, and the ORFs display signal-like sequences which canindicate that the described NHP ORFs are secreted proteins or can bemembrane associated.

[0054] The NHP amino acid sequences of the invention include the aminoacid sequences presented in the Sequence Listing as well as analoguesand derivatives thereof. Further, corresponding NHP homologues fromother species are encompassed by the invention. In fact, any NHPsencoded by a NHP nucleotide sequence described above are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code is wellknown, and, accordingly, each amino acid presented in the SequenceListing, is generically representative of the well known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al. eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference) are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

[0055] The invention also encompasses proteins that are functionallyequivalent to the NHPs encoded by the presently described nucleotidesequences as judged by any of a number of criteria, including, but notlimited to, the ability to bind and cleave a substrate of a NHP, or theability to effect an identical or complementary downstream pathway, or achange in cellular metabolism (e.g., proteolytic activity, ion flux,tyrosine phosphorylation, etc.). Such functionally equivalent NHPproteins include, but are not limited to, additions or substitutions ofamino acid residues within the amino acid sequence encoded by the NHPnucleotide sequences described above, but which result in a silentchange, thus producing a functionally equivalent expression product.

[0056] Amino acid substitutions can be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity, and/orthe amphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

[0057] A variety of host-expression vector systems can be used toexpress the NHP nucleotide sequences of the invention. Where, as in thepresent instance, a NHP peptide or NHP polypeptide is thought to be asoluble or secreted molecule, the peptide or polypeptide can berecovered from the culture media. Such expression systems also encompassengineered host cells that express NHP, or functional equivalent, insitu. Purification or enrichment of a NHP from such expression systemscan be accomplished using appropriate detergents and lipid micelles andmethods well known to those skilled in the art. However, such engineeredhost cells themselves may be used in situations where it is importantnot only to retain the structural and functional characteristics of aNHP, but to assess biological activity, e.g., in drug screening assays.

[0058] The expression systems that may be used for purposes of theinvention include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing NHPnucleotide sequences; yeast (e.g., Saccharomyces, Pichia) transformedwith recombinant yeast expression vectors containing NHP encodingnucleotide sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing NHP sequences;plant cell systems infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing NHP nucleotide sequences; or mammalian cell systems(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0059] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of and/or containing a NHP, or for raising antibodies to aNHP, vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited, to the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouye& Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Pharmacia or American Type Culture Collection) can also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The PGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target expression productcan be released from the GST moiety.

[0060] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign polynucleotidesequences. The virus grows in Spodoptera frugiperda cells. A NHP codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of NHP coding sequence will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted sequence is expressed (e.g., see Smith etal., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

[0061] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the NHP nucleotide sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericsequence may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing a NHP product in infected hosts(e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted NHP nucleotide sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire NHP gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of a NHP coding sequenceis inserted, exogenous translational control signals, including,perhaps, the ATG initiation codon, must be provided. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (See Bitter et al.,1987, Methods in Enzymol. 153:516-544).

[0062] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the correct modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells which possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

[0063] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the NHP sequences described above can be engineered. Rather thanusing expression vectors which contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell lineswhich express a NHP product. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds that affectthe endogenous activity of a NHP product.

[0064] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817)genes, which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for the following genes: dhfr, which confers resistance tomethotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567;O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, whichconfers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc.Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to theaminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol.150:1); and hygro, which confers resistance to hygromycin (Santerre, etal., 1984, Gene 30:147).

[0065] Alternatively, any fusion protein can be readily purified byutilizing an antibody specific for the fusion protein being expressed.For example, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the sequence of interest is subclonedinto a vaccinia recombination plasmid such that the sequence's openreading frame is translationally fused to an amino-terminal tagconsisting of six histidine residues. Extracts from cells infected withrecombinant vaccinia virus are loaded onto Ni²⁺nitriloaceticacid-agarose columns and histidine-tagged proteins are selectivelyeluted with imidazole-containing buffers.

[0066] Also encompassed by the present invention are fusion proteinsthat direct the NHP to a target organ and/or facilitate transport acrossthe membrane into the cytosol. Conjugation of NHPs to antibody moleculesor their Fab fragments could be used to target cells bearing aparticular epitope. Attaching the appropriate signal sequence to the NHPwould also transport the NHP to the desired location within the cell.Alternatively targeting of NHP or its nucleic acid sequence might beachieved using liposome or lipid complex based delivery systems. Suchtechnologies are described in “Liposomes:A Practical Approach” , New,R.R.C., ed., Oxford University Press, New York and in U.S. Pat. Nos.4,594,595, 5,459,127, 5,948,767 and 6,110,490 and their respectivedisclosures which are herein incorporated by reference in theirentirety. Additionally embodied are novel protein constructs engineeredin such a way that they facilitate transport of the NHP to the targetsite or desired organ, where they cross the cell membrane and/or thenucleus where the NHP can exert its functional activity. This goal maybe achieved by coupling of the NHP to a cytokine or other ligand thatprovides targeting specificity, and/or to a protein transducing domain(see generally U.S. applications Ser. No. 60/111,701 and 60/056,713,both of which are herein incorporated by reference, for examples of suchtransducing sequences) to facilitate passage across cellular membranesand can optionally be engineered to include nuclear localization.

5.3 Antibodies to Nhp Products

[0067] Antibodies that specifically recognize one or more epitopes of aNHP, or epitopes of conserved variants of a NHP, or peptide fragments ofa NHP are also encompassed by the invention. Such antibodies include butare not limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

[0068] The antibodies of the invention may be used, for example, in thedetection of a NHP in a biological sample and may, therefore, beutilized as part of a diagnostic or prognostic technique wherebypatients may be tested for abnormal amounts of NHP. Such antibodies mayalso be utilized in conjunction with, for example, compound screeningschemes for the evaluation of the effect of test compounds on expressionand/or activity of a NHP expression product. Additionally, suchantibodies can be used in conjunction gene therapy to, for example,evaluate the normal and/or engineered NHP-expressing cells prior totheir introduction into the patient. Such antibodies may additionally beused as a method for the inhibition of abnormal NHP activity. Thus, suchantibodies may, therefore, be utilized as part of treatment methods.

[0069] For the production of antibodies, various host animals may beimmunized by injection with a NHP, an NHP peptide (e.g., onecorresponding to a functional domain of a NHP), truncated NHPpolypeptides (NHP in which one or more domains have been deleted),functional equivalents of a NHP or mutated variants of a NHP. Such hostanimals may include but are not limited to pigs, rabbits, mice, goats,and rats, to name but a few. Various adjuvants may be used to increasethe immunological response, depending on the host species, including butnot limited to Freund's adjuvant (complete and incomplete), mineralsalts such as aluminum hydroxide or aluminum phosphate, chitosan,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Alternatively, the immune response could be enhanced bycombination and or coupling with molecules such as keyhole limpethemocyanin, tetanus toxoid, diphtheria toxoid, ovalbumin, cholera toxinor fragments thereof. Polyclonal antibodies are heterogeneouspopulations of antibody molecules derived from the sera of the immunizedanimals.

[0070] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mAbs in vivo makes this thepresently preferred method of production.

[0071] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused (see U.S. Pat. Nos. 5,877,397 and 6,075,181 herein incorporated byreference in their entirety). A chimeric antibody is a molecule in whichdifferent portions are derived from different animal species, such asthose having a variable region derived from a murine mAb and a humanimmunoglobulin constant region. Such technologies are described in U.S.Pat. Nos. 6,075,181 and 5,877,397 and their respective disclosures whichare herein incorporated by reference in their entirety. Also encompassedby the present invention is the use of fully humanized monoclonalantibodies as described in U.S. Pat. No. 6,150,584 and respectivedisclosures which are herein incorporated by reference in theirentirety.

[0072] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 341:544-546) can be adaptedto produce single chain antibodies against NHP expression products.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0073] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, such fragments include, butare not limited to: the F(ab′) ₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

[0074] Antibodies to a NHP can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” a given NHP, using techniques wellknown to those skilled in the art. (See, e.g., Greenspan & Bona, 1993,FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438). For example antibodies which bind to a NHP domain andcompetitively inhibit the binding of NHP to its cognate receptor can beused to generate anti-idiotypes that “mimic” a NHP and, therefore, bindand activate or neutralize a receptor. Such anti-idiotypic antibodies orFab fragments of such anti-idiotypes can be used in therapeutic regimensinvolving a NHP signaling pathway.

[0075] Additionally given the high degree of relatedness of mammalianNHPs, the presently described knock-out mice (having never seen NHP, andthus never been tolerized to NHP) have a unique utility, as they can beadvantageously applied to the generation of antibodies against thedisclosed mammalian NHP (i.e., NHP will be immunogenic in NHP knock-outanimals).

[0076] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description. Such modifications are intended tofall within the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

1 9 1 2781 DNA homo sapiens 1 atgccagggg gcgcaggcgc cgcccggctctgcttgctgg cgtttgccct gcagcccctc 60 cggccgcggg cggcgcggga gcctggatggacaagaggaa gtgaggaagg cagccccaag 120 ctgcagcatg aacttatcat acctcagtggaagacttcag aaagccccgt gagagaaaag 180 catccactca aagctgagct cagggtaatggctgaggggc gagaactgat cctggacctg 240 gagaagaatg agcaactttt tgctccttcctacacagaaa cccattatac ttcaagtggt 300 aaccctcaaa ccaccacacg gaaattggaggatcactgct tttaccacgg cacggtgagg 360 gagacagaac tgtccagcgt cacgctcagcacttgccgag gaattagagg actgattacg 420 gtgagcagca acctcagcta cgtcatcgagcccctccctg acagcaaggg ccaacacctt 480 atttacagat ctgaacatct caagccgcccccgggaaact gtgggttcga gcactccaag 540 cccaccacca gggactgggc tcttcagtttacacaacaga ccaagaagcg acctcgcagg 600 atgaaaaggg aagatttaaa ctccatgaagtatgtggagc tttacctcgt ggctgattat 660 ttagagtttc agaagaatcg acgagaccaggacgccacca aacacaagct catagagatc 720 gccaactatg ttgataagtt ttaccgatccttgaacatcc ggattgctct cgtgggcttg 780 gaagtgtgga cccacgggaa catgtgtgaagtttcagaga atccatattc taccctctgg 840 tcctttctca gttggaggcg caagctgcttgcccagaagt accatgacaa cgcccaatta 900 atcacgggca tgtccttcca cggcaccaccatcggcctgg cccccctcat ggccatgtgc 960 tctgtgtacc agtctggagg agtcaacatggaccactccg agaatgccat tggcgtggct 1020 gccaccatgg cccacgagat gggccacaactttggcatga cccatgattc tgcagattgc 1080 tgctcggcca gtgcggctga tggtgggtgcatcatggcag ctgccactgg gcaccccttt 1140 cccaaagtgt tcaatggatg caacaggagggagctggaca ggtatctgca gtcaggtggt 1200 ggaatgtgtc tctccaacat gccagacaccaggatgttgt atggaggccg gaggtgtggg 1260 aacgggtatc tggaagatgg ggaagagtgtgactgtggag aagaagagga atgtaacaac 1320 ccctgctgca atgcctctaa ttgtaccctgaggccggggg cggagtgtgc tcacggctcc 1380 tgctgccacc agtgtaagct gttggctcctgggaccctgt gccgcgagca ggccaggcag 1440 tgtgacctcc cggagttctg tacgggcaagtctccccact gccctaccaa cttctaccag 1500 atggatggta ccccctgtga gggcggccaggcctactgct acaacggcat gtgcctcacc 1560 taccaggagc agtgccagca gctgtggggacccggagccc gacctgcccc tgacctctgc 1620 ttcgagaagg tgaatgtggc aggagacacctttggaaact gtggaaagga catgaatggt 1680 gaacacagga agtgcaacat gagagatgcgaagtgtggga agatccagtg tcagagctct 1740 gaggcccggc ccctggagtc caacgcggtgcccattgaca ccactatcat catgaatggg 1800 aggcagatcc agtgccgggg cacccacgtctaccgaggtc ctgaggagga gggtgacatg 1860 ctggacccag ggctggtgat gactggaaccaagtgtggct acaaccatat ttgctttgag 1920 gggcagtgca ggaacacctc cttctttgaaactgaaggct gtgggaagaa gtgcaatggc 1980 catggggtct gtaacaacaa ccagaactgccactgcctgc cgggctgggc cccgcccttc 2040 tgcaacacac cgggccacgg gggcagtatcgacagtgggc ctatgccccc tgagagtgtg 2100 ggtcctgtgg tagctggagt gttggtggccatcttggtgc tggcggtcct catgctgatg 2160 tactactgct gcagacagaa caacaaactaggccaactca agccctcagc tctcccttcc 2220 aagctgaggc aacagttcag ttgtcccttcagggtttctc agaacagcgg gactggtcat 2280 gccaacccaa ctttcaagct gcagacgccccagggcaagc gaaaggtgtt ccttgacttg 2340 tgcgtacagg tgatcaacac tccggaaatcctgcggaagc cctcccagcc tcctccccgg 2400 ccccctccag attatctgcg tggtgggtccccacctgcac cactgccagc tcacctgagc 2460 agggctgcta ggaactcccc agggcccgggtctcaaatag agaggacgga gtcgtccagg 2520 aggcctcctc caagccggcc aattccccccgcaccaaatt gcatcgtttc ccaggacttc 2580 tccaggcctc ggccgcccca gaaggcactcccggcaaacc cagtgccagg ccgcaggagc 2640 ctccccaggc caggaggtgc atccccactgcggccccctg gtgctggccc tcagcagtcc 2700 cggcctctgg cagcacttgc cccaaagagggtatggaaga cttgcaattt gaaaactggg 2760 gaccagttcc aaagtcagta a 2781 2 926PRT homo sapiens 2 Met Pro Gly Gly Ala Gly Ala Ala Arg Leu Cys Leu LeuAla Phe Ala 1 5 10 15 Leu Gln Pro Leu Arg Pro Arg Ala Ala Arg Glu ProGly Trp Thr Arg 20 25 30 Gly Ser Glu Glu Gly Ser Pro Lys Leu Gln His GluLeu Ile Ile Pro 35 40 45 Gln Trp Lys Thr Ser Glu Ser Pro Val Arg Glu LysHis Pro Leu Lys 50 55 60 Ala Glu Leu Arg Val Met Ala Glu Gly Arg Glu LeuIle Leu Asp Leu 65 70 75 80 Glu Lys Asn Glu Gln Leu Phe Ala Pro Ser TyrThr Glu Thr His Tyr 85 90 95 Thr Ser Ser Gly Asn Pro Gln Thr Thr Thr ArgLys Leu Glu Asp His 100 105 110 Cys Phe Tyr His Gly Thr Val Arg Glu ThrGlu Leu Ser Ser Val Thr 115 120 125 Leu Ser Thr Cys Arg Gly Ile Arg GlyLeu Ile Thr Val Ser Ser Asn 130 135 140 Leu Ser Tyr Val Ile Glu Pro LeuPro Asp Ser Lys Gly Gln His Leu 145 150 155 160 Ile Tyr Arg Ser Glu HisLeu Lys Pro Pro Pro Gly Asn Cys Gly Phe 165 170 175 Glu His Ser Lys ProThr Thr Arg Asp Trp Ala Leu Gln Phe Thr Gln 180 185 190 Gln Thr Lys LysArg Pro Arg Arg Met Lys Arg Glu Asp Leu Asn Ser 195 200 205 Met Lys TyrVal Glu Leu Tyr Leu Val Ala Asp Tyr Leu Glu Phe Gln 210 215 220 Lys AsnArg Arg Asp Gln Asp Ala Thr Lys His Lys Leu Ile Glu Ile 225 230 235 240Ala Asn Tyr Val Asp Lys Phe Tyr Arg Ser Leu Asn Ile Arg Ile Ala 245 250255 Leu Val Gly Leu Glu Val Trp Thr His Gly Asn Met Cys Glu Val Ser 260265 270 Glu Asn Pro Tyr Ser Thr Leu Trp Ser Phe Leu Ser Trp Arg Arg Lys275 280 285 Leu Leu Ala Gln Lys Tyr His Asp Asn Ala Gln Leu Ile Thr GlyMet 290 295 300 Ser Phe His Gly Thr Thr Ile Gly Leu Ala Pro Leu Met AlaMet Cys 305 310 315 320 Ser Val Tyr Gln Ser Gly Gly Val Asn Met Asp HisSer Glu Asn Ala 325 330 335 Ile Gly Val Ala Ala Thr Met Ala His Glu MetGly His Asn Phe Gly 340 345 350 Met Thr His Asp Ser Ala Asp Cys Cys SerAla Ser Ala Ala Asp Gly 355 360 365 Gly Cys Ile Met Ala Ala Ala Thr GlyHis Pro Phe Pro Lys Val Phe 370 375 380 Asn Gly Cys Asn Arg Arg Glu LeuAsp Arg Tyr Leu Gln Ser Gly Gly 385 390 395 400 Gly Met Cys Leu Ser AsnMet Pro Asp Thr Arg Met Leu Tyr Gly Gly 405 410 415 Arg Arg Cys Gly AsnGly Tyr Leu Glu Asp Gly Glu Glu Cys Asp Cys 420 425 430 Gly Glu Glu GluGlu Cys Asn Asn Pro Cys Cys Asn Ala Ser Asn Cys 435 440 445 Thr Leu ArgPro Gly Ala Glu Cys Ala His Gly Ser Cys Cys His Gln 450 455 460 Cys LysLeu Leu Ala Pro Gly Thr Leu Cys Arg Glu Gln Ala Arg Gln 465 470 475 480Cys Asp Leu Pro Glu Phe Cys Thr Gly Lys Ser Pro His Cys Pro Thr 485 490495 Asn Phe Tyr Gln Met Asp Gly Thr Pro Cys Glu Gly Gly Gln Ala Tyr 500505 510 Cys Tyr Asn Gly Met Cys Leu Thr Tyr Gln Glu Gln Cys Gln Gln Leu515 520 525 Trp Gly Pro Gly Ala Arg Pro Ala Pro Asp Leu Cys Phe Glu LysVal 530 535 540 Asn Val Ala Gly Asp Thr Phe Gly Asn Cys Gly Lys Asp MetAsn Gly 545 550 555 560 Glu His Arg Lys Cys Asn Met Arg Asp Ala Lys CysGly Lys Ile Gln 565 570 575 Cys Gln Ser Ser Glu Ala Arg Pro Leu Glu SerAsn Ala Val Pro Ile 580 585 590 Asp Thr Thr Ile Ile Met Asn Gly Arg GlnIle Gln Cys Arg Gly Thr 595 600 605 His Val Tyr Arg Gly Pro Glu Glu GluGly Asp Met Leu Asp Pro Gly 610 615 620 Leu Val Met Thr Gly Thr Lys CysGly Tyr Asn His Ile Cys Phe Glu 625 630 635 640 Gly Gln Cys Arg Asn ThrSer Phe Phe Glu Thr Glu Gly Cys Gly Lys 645 650 655 Lys Cys Asn Gly HisGly Val Cys Asn Asn Asn Gln Asn Cys His Cys 660 665 670 Leu Pro Gly TrpAla Pro Pro Phe Cys Asn Thr Pro Gly His Gly Gly 675 680 685 Ser Ile AspSer Gly Pro Met Pro Pro Glu Ser Val Gly Pro Val Val 690 695 700 Ala GlyVal Leu Val Ala Ile Leu Val Leu Ala Val Leu Met Leu Met 705 710 715 720Tyr Tyr Cys Cys Arg Gln Asn Asn Lys Leu Gly Gln Leu Lys Pro Ser 725 730735 Ala Leu Pro Ser Lys Leu Arg Gln Gln Phe Ser Cys Pro Phe Arg Val 740745 750 Ser Gln Asn Ser Gly Thr Gly His Ala Asn Pro Thr Phe Lys Leu Gln755 760 765 Thr Pro Gln Gly Lys Arg Lys Val Phe Leu Asp Leu Cys Val GlnVal 770 775 780 Ile Asn Thr Pro Glu Ile Leu Arg Lys Pro Ser Gln Pro ProPro Arg 785 790 795 800 Pro Pro Pro Asp Tyr Leu Arg Gly Gly Ser Pro ProAla Pro Leu Pro 805 810 815 Ala His Leu Ser Arg Ala Ala Arg Asn Ser ProGly Pro Gly Ser Gln 820 825 830 Ile Glu Arg Thr Glu Ser Ser Arg Arg ProPro Pro Ser Arg Pro Ile 835 840 845 Pro Pro Ala Pro Asn Cys Ile Val SerGln Asp Phe Ser Arg Pro Arg 850 855 860 Pro Pro Gln Lys Ala Leu Pro AlaAsn Pro Val Pro Gly Arg Arg Ser 865 870 875 880 Leu Pro Arg Pro Gly GlyAla Ser Pro Leu Arg Pro Pro Gly Ala Gly 885 890 895 Pro Gln Gln Ser ArgPro Leu Ala Ala Leu Ala Pro Lys Arg Val Trp 900 905 910 Lys Thr Cys AsnLeu Lys Thr Gly Asp Gln Phe Gln Ser Gln 915 920 925 3 2757 DNA homosapiens 3 atgccagggg gcgcaggcgc cgcccggctc tgcttgctgg cgtttgccctgcagcccctc 60 cggccgcggg cggcgcggga gcctggatgg acaagaggaa gtgaggaaggcagccccaag 120 ctgcagcatg aacttatcat acctcagtgg aagacttcag aaagccccgtgagagaaaag 180 catccactca aagctgagct cagggtaatg gctgaggggc gagaactgatcctggacctg 240 gagaagaatg agcaactttt tgctccttcc tacacagaaa cccattatacttcaagtggt 300 aaccctcaaa ccaccacacg gaaattggag gatcactgct tttaccacggcacggtgagg 360 gagacagaac tgtccagcgt cacgctcagc acttgccgag gaattagaggactgattacg 420 gtgagcagca acctcagcta cgtcatcgag cccctccctg acagcaagggccaacacctt 480 atttacagat ctgaacatct caagccgccc ccgggaaact gtgggttcgagcactccaag 540 cccaccacca gggactgggc tcttcagttt acacaacaga ccaagaagcgacctcgcagg 600 atgaaaaggg aagatttaaa ctccatgaag tatgtggagc tttacctcgtggctgattat 660 ttagagtttc agaagaatcg acgagaccag gacgccacca aacacaagctcatagagatc 720 gccaactatg ttgataagtt ttaccgatcc ttgaacatcc ggattgctctcgtgggcttg 780 gaagtgtgga cccacgggaa catgtgtgaa gtttcagaga atccatattctaccctctgg 840 tcctttctca gttggaggcg caagctgctt gcccagaagt accatgacaacgcccaatta 900 atcacgggca tgtccttcca cggcaccacc atcggcctgg cccccctcatggccatgtgc 960 tctgtgtacc agtctggagg agtcaacatg gaccactccg agaatgccattggcgtggct 1020 gccaccatgg cccacgagat gggccacaac tttggcatga cccatgattctgcagattgc 1080 tgctcggcca gtgcggctga tggtgggtgc atcatggcag ctgccactgggcaccccttt 1140 cccaaagtgt tcaatggatg caacaggagg gagctggaca ggtatctgcagtcaggtggt 1200 ggaatgtgtc tctccaacat gccagacacc aggatgttgt atggaggccggaggtgtggg 1260 aacgggtatc tggaagatgg ggaagagtgt gactgtggag aagaagaggaatgtaacaac 1320 ccctgctgca atgcctctaa ttgtaccctg aggccggggg cggagtgtgctcacggctcc 1380 tgctgccacc agtgtaagct gttggctcct gggaccctgt gccgcgagcaggccaggcag 1440 tgtgacctcc cggagttctg tacgggcaag tctccccact gccctaccaacttctaccag 1500 atggatggta ccccctgtga gggcggccag gcctactgct acaacggcatgtgcctcacc 1560 taccaggagc agtgccagca gctgtgggga cccggagccc gacctgcccctgacctctgc 1620 ttcgagaagg tgaatgtggc aggagacacc tttggaaact gtggaaaggacatgaatggt 1680 gaacacagga agtgcaacat gagagatgcg aagtgtggga agatccagtgtcagagctct 1740 gaggcccggc ccctggagtc caacgcggtg cccattgaca ccactatcatcatgaatggg 1800 aggcagatcc agtgccgggg cacccacgtc taccgaggtc ctgaggaggagggtgacatg 1860 ctggacccag ggctggtgat gactggaacc aagtgtggct acaaccatatttgctttgag 1920 gggcagtgca ggaacacctc cttctttgaa actgaaggct gtgggaagaagtgcaatggc 1980 catggggtct gtaacaacaa ccagaactgc cactgcctgc cgggctgggccccgcccttc 2040 tgcaacacac cgggccacgg gggcagtatc gacagtgggc ctatgccccctgagagtgtg 2100 ggtcctgtgg tagctggagt gttggtggcc atcttggtgc tggcggtcctcatgctgatg 2160 tactactgct gcagacagaa caacaaacta ggccaactca agccctcagctctcccttcc 2220 aagctgaggc aacagttcag ttgtcccttc agggtttctc agaacagcgggactggtcat 2280 gccaacccaa ctttcaagct gcagacgccc cagggcaagc gaaaggtgatcaacactccg 2340 gaaatcctgc ggaagccctc ccagcctcct ccccggcccc ctccagattatctgcgtggt 2400 gggtccccac ctgcaccact gccagctcac ctgagcaggg ctgctaggaactccccaggg 2460 cccgggtctc aaatagagag gacggagtcg tccaggaggc ctcctccaagccggccaatt 2520 ccccccgcac caaattgcat cgtttcccag gacttctcca ggcctcggccgccccagaag 2580 gcactcccgg caaacccagt gccaggccgc aggagcctcc ccaggccaggaggtgcatcc 2640 ccactgcggc cccctggtgc tggccctcag cagtcccggc ctctggcagcacttgcccca 2700 aagagggtat ggaagacttg caatttgaaa actggggacc agttccaaagtcagtaa 2757 4 918 PRT homo sapiens 4 Met Pro Gly Gly Ala Gly Ala AlaArg Leu Cys Leu Leu Ala Phe Ala 1 5 10 15 Leu Gln Pro Leu Arg Pro ArgAla Ala Arg Glu Pro Gly Trp Thr Arg 20 25 30 Gly Ser Glu Glu Gly Ser ProLys Leu Gln His Glu Leu Ile Ile Pro 35 40 45 Gln Trp Lys Thr Ser Glu SerPro Val Arg Glu Lys His Pro Leu Lys 50 55 60 Ala Glu Leu Arg Val Met AlaGlu Gly Arg Glu Leu Ile Leu Asp Leu 65 70 75 80 Glu Lys Asn Glu Gln LeuPhe Ala Pro Ser Tyr Thr Glu Thr His Tyr 85 90 95 Thr Ser Ser Gly Asn ProGln Thr Thr Thr Arg Lys Leu Glu Asp His 100 105 110 Cys Phe Tyr His GlyThr Val Arg Glu Thr Glu Leu Ser Ser Val Thr 115 120 125 Leu Ser Thr CysArg Gly Ile Arg Gly Leu Ile Thr Val Ser Ser Asn 130 135 140 Leu Ser TyrVal Ile Glu Pro Leu Pro Asp Ser Lys Gly Gln His Leu 145 150 155 160 IleTyr Arg Ser Glu His Leu Lys Pro Pro Pro Gly Asn Cys Gly Phe 165 170 175Glu His Ser Lys Pro Thr Thr Arg Asp Trp Ala Leu Gln Phe Thr Gln 180 185190 Gln Thr Lys Lys Arg Pro Arg Arg Met Lys Arg Glu Asp Leu Asn Ser 195200 205 Met Lys Tyr Val Glu Leu Tyr Leu Val Ala Asp Tyr Leu Glu Phe Gln210 215 220 Lys Asn Arg Arg Asp Gln Asp Ala Thr Lys His Lys Leu Ile GluIle 225 230 235 240 Ala Asn Tyr Val Asp Lys Phe Tyr Arg Ser Leu Asn IleArg Ile Ala 245 250 255 Leu Val Gly Leu Glu Val Trp Thr His Gly Asn MetCys Glu Val Ser 260 265 270 Glu Asn Pro Tyr Ser Thr Leu Trp Ser Phe LeuSer Trp Arg Arg Lys 275 280 285 Leu Leu Ala Gln Lys Tyr His Asp Asn AlaGln Leu Ile Thr Gly Met 290 295 300 Ser Phe His Gly Thr Thr Ile Gly LeuAla Pro Leu Met Ala Met Cys 305 310 315 320 Ser Val Tyr Gln Ser Gly GlyVal Asn Met Asp His Ser Glu Asn Ala 325 330 335 Ile Gly Val Ala Ala ThrMet Ala His Glu Met Gly His Asn Phe Gly 340 345 350 Met Thr His Asp SerAla Asp Cys Cys Ser Ala Ser Ala Ala Asp Gly 355 360 365 Gly Cys Ile MetAla Ala Ala Thr Gly His Pro Phe Pro Lys Val Phe 370 375 380 Asn Gly CysAsn Arg Arg Glu Leu Asp Arg Tyr Leu Gln Ser Gly Gly 385 390 395 400 GlyMet Cys Leu Ser Asn Met Pro Asp Thr Arg Met Leu Tyr Gly Gly 405 410 415Arg Arg Cys Gly Asn Gly Tyr Leu Glu Asp Gly Glu Glu Cys Asp Cys 420 425430 Gly Glu Glu Glu Glu Cys Asn Asn Pro Cys Cys Asn Ala Ser Asn Cys 435440 445 Thr Leu Arg Pro Gly Ala Glu Cys Ala His Gly Ser Cys Cys His Gln450 455 460 Cys Lys Leu Leu Ala Pro Gly Thr Leu Cys Arg Glu Gln Ala ArgGln 465 470 475 480 Cys Asp Leu Pro Glu Phe Cys Thr Gly Lys Ser Pro HisCys Pro Thr 485 490 495 Asn Phe Tyr Gln Met Asp Gly Thr Pro Cys Glu GlyGly Gln Ala Tyr 500 505 510 Cys Tyr Asn Gly Met Cys Leu Thr Tyr Gln GluGln Cys Gln Gln Leu 515 520 525 Trp Gly Pro Gly Ala Arg Pro Ala Pro AspLeu Cys Phe Glu Lys Val 530 535 540 Asn Val Ala Gly Asp Thr Phe Gly AsnCys Gly Lys Asp Met Asn Gly 545 550 555 560 Glu His Arg Lys Cys Asn MetArg Asp Ala Lys Cys Gly Lys Ile Gln 565 570 575 Cys Gln Ser Ser Glu AlaArg Pro Leu Glu Ser Asn Ala Val Pro Ile 580 585 590 Asp Thr Thr Ile IleMet Asn Gly Arg Gln Ile Gln Cys Arg Gly Thr 595 600 605 His Val Tyr ArgGly Pro Glu Glu Glu Gly Asp Met Leu Asp Pro Gly 610 615 620 Leu Val MetThr Gly Thr Lys Cys Gly Tyr Asn His Ile Cys Phe Glu 625 630 635 640 GlyGln Cys Arg Asn Thr Ser Phe Phe Glu Thr Glu Gly Cys Gly Lys 645 650 655Lys Cys Asn Gly His Gly Val Cys Asn Asn Asn Gln Asn Cys His Cys 660 665670 Leu Pro Gly Trp Ala Pro Pro Phe Cys Asn Thr Pro Gly His Gly Gly 675680 685 Ser Ile Asp Ser Gly Pro Met Pro Pro Glu Ser Val Gly Pro Val Val690 695 700 Ala Gly Val Leu Val Ala Ile Leu Val Leu Ala Val Leu Met LeuMet 705 710 715 720 Tyr Tyr Cys Cys Arg Gln Asn Asn Lys Leu Gly Gln LeuLys Pro Ser 725 730 735 Ala Leu Pro Ser Lys Leu Arg Gln Gln Phe Ser CysPro Phe Arg Val 740 745 750 Ser Gln Asn Ser Gly Thr Gly His Ala Asn ProThr Phe Lys Leu Gln 755 760 765 Thr Pro Gln Gly Lys Arg Lys Val Ile AsnThr Pro Glu Ile Leu Arg 770 775 780 Lys Pro Ser Gln Pro Pro Pro Arg ProPro Pro Asp Tyr Leu Arg Gly 785 790 795 800 Gly Ser Pro Pro Ala Pro LeuPro Ala His Leu Ser Arg Ala Ala Arg 805 810 815 Asn Ser Pro Gly Pro GlySer Gln Ile Glu Arg Thr Glu Ser Ser Arg 820 825 830 Arg Pro Pro Pro SerArg Pro Ile Pro Pro Ala Pro Asn Cys Ile Val 835 840 845 Ser Gln Asp PheSer Arg Pro Arg Pro Pro Gln Lys Ala Leu Pro Ala 850 855 860 Asn Pro ValPro Gly Arg Arg Ser Leu Pro Arg Pro Gly Gly Ala Ser 865 870 875 880 ProLeu Arg Pro Pro Gly Ala Gly Pro Gln Gln Ser Arg Pro Leu Ala 885 890 895Ala Leu Ala Pro Lys Arg Val Trp Lys Thr Cys Asn Leu Lys Thr Gly 900 905910 Asp Gln Phe Gln Ser Gln 915 5 2892 DNA homo sapiens 5 atgccagggggcgcaggcgc cgcccggctc tgcttgctgg cgtttgccct gcagcccctc 60 cggccgcgggcggcgcggga gcctggatgg acaagaggaa gtgaggaagg cagccccaag 120 ctgcagcatgaacttatcat acctcagtgg aagacttcag aaagccccgt gagagaaaag 180 catccactcaaagctgagct cagggtaatg gctgaggggc gagaactgat cctggacctg 240 gagaagaatgagcaactttt tgctccttcc tacacagaaa cccattatac ttcaagtggt 300 aaccctcaaaccaccacacg gaaattggag gatcactgct tttaccacgg cacggtgagg 360 gagacagaactgtccagcgt cacgctcagc acttgccgag gaattagagg actgattacg 420 gtgagcagcaacctcagcta cgtcatcgag cccctccctg acagcaaggg ccaacacctt 480 atttacagatctgaacatct caagccgccc ccgggaaact gtgggttcga gcactccaag 540 cccaccaccagggactgggc tcttcagttt acacaacaga ccaagaagcg acctcgcagg 600 atgaaaagggaagatttaaa ctccatgaag tatgtggagc tttacctcgt ggctgattat 660 ttagagtttcagaagaatcg acgagaccag gacgccacca aacacaagct catagagatc 720 gccaactatgttgataagtt ttaccgatcc ttgaacatcc ggattgctct cgtgggcttg 780 gaagtgtggacccacgggaa catgtgtgaa gtttcagaga atccatattc taccctctgg 840 tcctttctcagttggaggcg caagctgctt gcccagaagt accatgacaa cgcccaatta 900 atcacgggcatgtccttcca cggcaccacc atcggcctgg cccccctcat ggccatgtgc 960 tctgtgtaccagtctggagg agtcaacatg gaccactccg agaatgccat tggcgtggct 1020 gccaccatggcccacgagat gggccacaac tttggcatga cccatgattc tgcagattgc 1080 tgctcggccagtgcggctga tggtgggtgc atcatggcag ctgccactgg gcaccccttt 1140 cccaaagtgttcaatggatg caacaggagg gagctggaca ggtatctgca gtcaggtggt 1200 ggaatgtgtctctccaacat gccagacacc aggatgttgt atggaggccg gaggtgtggg 1260 aacgggtatctggaagatgg ggaagagtgt gactgtggag aagaagagga atgtaacaac 1320 ccctgctgcaatgcctctaa ttgtaccctg aggccggggg cggagtgtgc tcacggctcc 1380 tgctgccaccagtgtaagct gttggctcct gggaccctgt gccgcgagca ggccaggcag 1440 tgtgacctcccggagttctg tacgggcaag tctccccact gccctaccaa cttctaccag 1500 atggatggtaccccctgtga gggcggccag gcctactgct acaacggcat gtgcctcacc 1560 taccaggagcagtgccagca gctgtgggga cccggagccc gacctgcccc tgacctctgc 1620 ttcgagaaggtgaatgtggc aggagacacc tttggaaact gtggaaagga catgaatggt 1680 gaacacaggaagtgcaacat gagagatgcg aagtgtggga agatccagtg tcagagctct 1740 gaggcccggcccctggagtc caacgcggtg cccattgaca ccactatcat catgaatggg 1800 aggcagatccagtgccgggg cacccacgtc taccgaggtc ctgaggagga gggtgacatg 1860 ctggacccagggctggtgat gactggaacc aagtgtggct acaaccatat ttgctttgag 1920 gggcagtgcaggaacacctc cttctttgaa actgaaggct gtgggaagaa gtgcaatggc 1980 catggggtctgtaacaacaa ccagaactgc cactgcctgc cgggctgggc cccgcccttc 2040 tgcaacacaccgggccacgg gggcagtatc gacagtgggc ctatgccccc tgagagtgtg 2100 ggtcctgtggtagctggagt gttggtggcc atcttggtgc tggcggtcct catgctgatg 2160 tactactgctgcagacagaa caacaaacta ggccaactca agccctcagc tctcccttcc 2220 aagctgaggcaacagttcag ttgtcccttc agggtttctc agaacagcgg gactggtcat 2280 gccaacccaactttcaagct gcagacgccc cagggcaagc gaaaggtgtt ccttgacttg 2340 tgcgtacaggtgatcaacac tccggaaatc ctgcggaagc cctcccagcc tcctccccgg 2400 ccccctccagattatctgcg tggtgggtcc ccacctgcac cactgccagc tcacctgagc 2460 agggctgctaggaactcccc agggcccggg tctcaaatag agaggacgga gtcgtccagg 2520 aggcctcctccaagccggcc aattcccccc gcaccaaatt gcatcgtttc ccaggacttc 2580 tccaggcctcggccgcccca gaaggcactc ccggcaaacc cagtgccagg ccgcaggagc 2640 ctccccaggccaggaggtgc atccccactg cggccccctg gtgctggccc tcagcagtcc 2700 cggcctctggcagcacttgc cccaaaggtg agtccacggg aagccctcaa ggtgaaagct 2760 ggtaccagagggctccaggg gggcaggtgt agagttgaga aaacaaagca attcatgctt 2820 cttgtggtctggactgaact tccagaacaa aagccaaggg caaaacattc atgtttcttg 2880 gtgcccgcttga 2892 6 963 PRT homo sapiens 6 Met Pro Gly Gly Ala Gly Ala Ala Arg LeuCys Leu Leu Ala Phe Ala 1 5 10 15 Leu Gln Pro Leu Arg Pro Arg Ala AlaArg Glu Pro Gly Trp Thr Arg 20 25 30 Gly Ser Glu Glu Gly Ser Pro Lys LeuGln His Glu Leu Ile Ile Pro 35 40 45 Gln Trp Lys Thr Ser Glu Ser Pro ValArg Glu Lys His Pro Leu Lys 50 55 60 Ala Glu Leu Arg Val Met Ala Glu GlyArg Glu Leu Ile Leu Asp Leu 65 70 75 80 Glu Lys Asn Glu Gln Leu Phe AlaPro Ser Tyr Thr Glu Thr His Tyr 85 90 95 Thr Ser Ser Gly Asn Pro Gln ThrThr Thr Arg Lys Leu Glu Asp His 100 105 110 Cys Phe Tyr His Gly Thr ValArg Glu Thr Glu Leu Ser Ser Val Thr 115 120 125 Leu Ser Thr Cys Arg GlyIle Arg Gly Leu Ile Thr Val Ser Ser Asn 130 135 140 Leu Ser Tyr Val IleGlu Pro Leu Pro Asp Ser Lys Gly Gln His Leu 145 150 155 160 Ile Tyr ArgSer Glu His Leu Lys Pro Pro Pro Gly Asn Cys Gly Phe 165 170 175 Glu HisSer Lys Pro Thr Thr Arg Asp Trp Ala Leu Gln Phe Thr Gln 180 185 190 GlnThr Lys Lys Arg Pro Arg Arg Met Lys Arg Glu Asp Leu Asn Ser 195 200 205Met Lys Tyr Val Glu Leu Tyr Leu Val Ala Asp Tyr Leu Glu Phe Gln 210 215220 Lys Asn Arg Arg Asp Gln Asp Ala Thr Lys His Lys Leu Ile Glu Ile 225230 235 240 Ala Asn Tyr Val Asp Lys Phe Tyr Arg Ser Leu Asn Ile Arg IleAla 245 250 255 Leu Val Gly Leu Glu Val Trp Thr His Gly Asn Met Cys GluVal Ser 260 265 270 Glu Asn Pro Tyr Ser Thr Leu Trp Ser Phe Leu Ser TrpArg Arg Lys 275 280 285 Leu Leu Ala Gln Lys Tyr His Asp Asn Ala Gln LeuIle Thr Gly Met 290 295 300 Ser Phe His Gly Thr Thr Ile Gly Leu Ala ProLeu Met Ala Met Cys 305 310 315 320 Ser Val Tyr Gln Ser Gly Gly Val AsnMet Asp His Ser Glu Asn Ala 325 330 335 Ile Gly Val Ala Ala Thr Met AlaHis Glu Met Gly His Asn Phe Gly 340 345 350 Met Thr His Asp Ser Ala AspCys Cys Ser Ala Ser Ala Ala Asp Gly 355 360 365 Gly Cys Ile Met Ala AlaAla Thr Gly His Pro Phe Pro Lys Val Phe 370 375 380 Asn Gly Cys Asn ArgArg Glu Leu Asp Arg Tyr Leu Gln Ser Gly Gly 385 390 395 400 Gly Met CysLeu Ser Asn Met Pro Asp Thr Arg Met Leu Tyr Gly Gly 405 410 415 Arg ArgCys Gly Asn Gly Tyr Leu Glu Asp Gly Glu Glu Cys Asp Cys 420 425 430 GlyGlu Glu Glu Glu Cys Asn Asn Pro Cys Cys Asn Ala Ser Asn Cys 435 440 445Thr Leu Arg Pro Gly Ala Glu Cys Ala His Gly Ser Cys Cys His Gln 450 455460 Cys Lys Leu Leu Ala Pro Gly Thr Leu Cys Arg Glu Gln Ala Arg Gln 465470 475 480 Cys Asp Leu Pro Glu Phe Cys Thr Gly Lys Ser Pro His Cys ProThr 485 490 495 Asn Phe Tyr Gln Met Asp Gly Thr Pro Cys Glu Gly Gly GlnAla Tyr 500 505 510 Cys Tyr Asn Gly Met Cys Leu Thr Tyr Gln Glu Gln CysGln Gln Leu 515 520 525 Trp Gly Pro Gly Ala Arg Pro Ala Pro Asp Leu CysPhe Glu Lys Val 530 535 540 Asn Val Ala Gly Asp Thr Phe Gly Asn Cys GlyLys Asp Met Asn Gly 545 550 555 560 Glu His Arg Lys Cys Asn Met Arg AspAla Lys Cys Gly Lys Ile Gln 565 570 575 Cys Gln Ser Ser Glu Ala Arg ProLeu Glu Ser Asn Ala Val Pro Ile 580 585 590 Asp Thr Thr Ile Ile Met AsnGly Arg Gln Ile Gln Cys Arg Gly Thr 595 600 605 His Val Tyr Arg Gly ProGlu Glu Glu Gly Asp Met Leu Asp Pro Gly 610 615 620 Leu Val Met Thr GlyThr Lys Cys Gly Tyr Asn His Ile Cys Phe Glu 625 630 635 640 Gly Gln CysArg Asn Thr Ser Phe Phe Glu Thr Glu Gly Cys Gly Lys 645 650 655 Lys CysAsn Gly His Gly Val Cys Asn Asn Asn Gln Asn Cys His Cys 660 665 670 LeuPro Gly Trp Ala Pro Pro Phe Cys Asn Thr Pro Gly His Gly Gly 675 680 685Ser Ile Asp Ser Gly Pro Met Pro Pro Glu Ser Val Gly Pro Val Val 690 695700 Ala Gly Val Leu Val Ala Ile Leu Val Leu Ala Val Leu Met Leu Met 705710 715 720 Tyr Tyr Cys Cys Arg Gln Asn Asn Lys Leu Gly Gln Leu Lys ProSer 725 730 735 Ala Leu Pro Ser Lys Leu Arg Gln Gln Phe Ser Cys Pro PheArg Val 740 745 750 Ser Gln Asn Ser Gly Thr Gly His Ala Asn Pro Thr PheLys Leu Gln 755 760 765 Thr Pro Gln Gly Lys Arg Lys Val Phe Leu Asp LeuCys Val Gln Val 770 775 780 Ile Asn Thr Pro Glu Ile Leu Arg Lys Pro SerGln Pro Pro Pro Arg 785 790 795 800 Pro Pro Pro Asp Tyr Leu Arg Gly GlySer Pro Pro Ala Pro Leu Pro 805 810 815 Ala His Leu Ser Arg Ala Ala ArgAsn Ser Pro Gly Pro Gly Ser Gln 820 825 830 Ile Glu Arg Thr Glu Ser SerArg Arg Pro Pro Pro Ser Arg Pro Ile 835 840 845 Pro Pro Ala Pro Asn CysIle Val Ser Gln Asp Phe Ser Arg Pro Arg 850 855 860 Pro Pro Gln Lys AlaLeu Pro Ala Asn Pro Val Pro Gly Arg Arg Ser 865 870 875 880 Leu Pro ArgPro Gly Gly Ala Ser Pro Leu Arg Pro Pro Gly Ala Gly 885 890 895 Pro GlnGln Ser Arg Pro Leu Ala Ala Leu Ala Pro Lys Val Ser Pro 900 905 910 ArgGlu Ala Leu Lys Val Lys Ala Gly Thr Arg Gly Leu Gln Gly Gly 915 920 925Arg Cys Arg Val Glu Lys Thr Lys Gln Phe Met Leu Leu Val Val Trp 930 935940 Thr Glu Leu Pro Glu Gln Lys Pro Arg Ala Lys His Ser Cys Phe Leu 945950 955 960 7 2868 DNA homo sapiens 7 atgccagggg gcgcaggcgc cgcccggctctgcttgctgg cgtttgccct gcagcccctc 60 cggccgcggg cggcgcggga gcctggatggacaagaggaa gtgaggaagg cagccccaag 120 ctgcagcatg aacttatcat acctcagtggaagacttcag aaagccccgt gagagaaaag 180 catccactca aagctgagct cagggtaatggctgaggggc gagaactgat cctggacctg 240 gagaagaatg agcaactttt tgctccttcctacacagaaa cccattatac ttcaagtggt 300 aaccctcaaa ccaccacacg gaaattggaggatcactgct tttaccacgg cacggtgagg 360 gagacagaac tgtccagcgt cacgctcagcacttgccgag gaattagagg actgattacg 420 gtgagcagca acctcagcta cgtcatcgagcccctccctg acagcaaggg ccaacacctt 480 atttacagat ctgaacatct caagccgcccccgggaaact gtgggttcga gcactccaag 540 cccaccacca gggactgggc tcttcagtttacacaacaga ccaagaagcg acctcgcagg 600 atgaaaaggg aagatttaaa ctccatgaagtatgtggagc tttacctcgt ggctgattat 660 ttagagtttc agaagaatcg acgagaccaggacgccacca aacacaagct catagagatc 720 gccaactatg ttgataagtt ttaccgatccttgaacatcc ggattgctct cgtgggcttg 780 gaagtgtgga cccacgggaa catgtgtgaagtttcagaga atccatattc taccctctgg 840 tcctttctca gttggaggcg caagctgcttgcccagaagt accatgacaa cgcccaatta 900 atcacgggca tgtccttcca cggcaccaccatcggcctgg cccccctcat ggccatgtgc 960 tctgtgtacc agtctggagg agtcaacatggaccactccg agaatgccat tggcgtggct 1020 gccaccatgg cccacgagat gggccacaactttggcatga cccatgattc tgcagattgc 1080 tgctcggcca gtgcggctga tggtgggtgcatcatggcag ctgccactgg gcaccccttt 1140 cccaaagtgt tcaatggatg caacaggagggagctggaca ggtatctgca gtcaggtggt 1200 ggaatgtgtc tctccaacat gccagacaccaggatgttgt atggaggccg gaggtgtggg 1260 aacgggtatc tggaagatgg ggaagagtgtgactgtggag aagaagagga atgtaacaac 1320 ccctgctgca atgcctctaa ttgtaccctgaggccggggg cggagtgtgc tcacggctcc 1380 tgctgccacc agtgtaagct gttggctcctgggaccctgt gccgcgagca ggccaggcag 1440 tgtgacctcc cggagttctg tacgggcaagtctccccact gccctaccaa cttctaccag 1500 atggatggta ccccctgtga gggcggccaggcctactgct acaacggcat gtgcctcacc 1560 taccaggagc agtgccagca gctgtggggacccggagccc gacctgcccc tgacctctgc 1620 ttcgagaagg tgaatgtggc aggagacacctttggaaact gtggaaagga catgaatggt 1680 gaacacagga agtgcaacat gagagatgcgaagtgtggga agatccagtg tcagagctct 1740 gaggcccggc ccctggagtc caacgcggtgcccattgaca ccactatcat catgaatggg 1800 aggcagatcc agtgccgggg cacccacgtctaccgaggtc ctgaggagga gggtgacatg 1860 ctggacccag ggctggtgat gactggaaccaagtgtggct acaaccatat ttgctttgag 1920 gggcagtgca ggaacacctc cttctttgaaactgaaggct gtgggaagaa gtgcaatggc 1980 catggggtct gtaacaacaa ccagaactgccactgcctgc cgggctgggc cccgcccttc 2040 tgcaacacac cgggccacgg gggcagtatcgacagtgggc ctatgccccc tgagagtgtg 2100 ggtcctgtgg tagctggagt gttggtggccatcttggtgc tggcggtcct catgctgatg 2160 tactactgct gcagacagaa caacaaactaggccaactca agccctcagc tctcccttcc 2220 aagctgaggc aacagttcag ttgtcccttcagggtttctc agaacagcgg gactggtcat 2280 gccaacccaa ctttcaagct gcagacgccccagggcaagc gaaaggtgat caacactccg 2340 gaaatcctgc ggaagccctc ccagcctcctccccggcccc ctccagatta tctgcgtggt 2400 gggtccccac ctgcaccact gccagctcacctgagcaggg ctgctaggaa ctccccaggg 2460 cccgggtctc aaatagagag gacggagtcgtccaggaggc ctcctccaag ccggccaatt 2520 ccccccgcac caaattgcat cgtttcccaggacttctcca ggcctcggcc gccccagaag 2580 gcactcccgg caaacccagt gccaggccgcaggagcctcc ccaggccagg aggtgcatcc 2640 ccactgcggc cccctggtgc tggccctcagcagtcccggc ctctggcagc acttgcccca 2700 aaggtgagtc cacgggaagc cctcaaggtgaaagctggta ccagagggct ccaggggggc 2760 aggtgtagag ttgagaaaac aaagcaattcatgcttcttg tggtctggac tgaacttcca 2820 gaacaaaagc caagggcaaa acattcatgtttcttggtgc ccgcttga 2868 8 955 PRT homo sapiens 8 Met Pro Gly Gly AlaGly Ala Ala Arg Leu Cys Leu Leu Ala Phe Ala 1 5 10 15 Leu Gln Pro LeuArg Pro Arg Ala Ala Arg Glu Pro Gly Trp Thr Arg 20 25 30 Gly Ser Glu GluGly Ser Pro Lys Leu Gln His Glu Leu Ile Ile Pro 35 40 45 Gln Trp Lys ThrSer Glu Ser Pro Val Arg Glu Lys His Pro Leu Lys 50 55 60 Ala Glu Leu ArgVal Met Ala Glu Gly Arg Glu Leu Ile Leu Asp Leu 65 70 75 80 Glu Lys AsnGlu Gln Leu Phe Ala Pro Ser Tyr Thr Glu Thr His Tyr 85 90 95 Thr Ser SerGly Asn Pro Gln Thr Thr Thr Arg Lys Leu Glu Asp His 100 105 110 Cys PheTyr His Gly Thr Val Arg Glu Thr Glu Leu Ser Ser Val Thr 115 120 125 LeuSer Thr Cys Arg Gly Ile Arg Gly Leu Ile Thr Val Ser Ser Asn 130 135 140Leu Ser Tyr Val Ile Glu Pro Leu Pro Asp Ser Lys Gly Gln His Leu 145 150155 160 Ile Tyr Arg Ser Glu His Leu Lys Pro Pro Pro Gly Asn Cys Gly Phe165 170 175 Glu His Ser Lys Pro Thr Thr Arg Asp Trp Ala Leu Gln Phe ThrGln 180 185 190 Gln Thr Lys Lys Arg Pro Arg Arg Met Lys Arg Glu Asp LeuAsn Ser 195 200 205 Met Lys Tyr Val Glu Leu Tyr Leu Val Ala Asp Tyr LeuGlu Phe Gln 210 215 220 Lys Asn Arg Arg Asp Gln Asp Ala Thr Lys His LysLeu Ile Glu Ile 225 230 235 240 Ala Asn Tyr Val Asp Lys Phe Tyr Arg SerLeu Asn Ile Arg Ile Ala 245 250 255 Leu Val Gly Leu Glu Val Trp Thr HisGly Asn Met Cys Glu Val Ser 260 265 270 Glu Asn Pro Tyr Ser Thr Leu TrpSer Phe Leu Ser Trp Arg Arg Lys 275 280 285 Leu Leu Ala Gln Lys Tyr HisAsp Asn Ala Gln Leu Ile Thr Gly Met 290 295 300 Ser Phe His Gly Thr ThrIle Gly Leu Ala Pro Leu Met Ala Met Cys 305 310 315 320 Ser Val Tyr GlnSer Gly Gly Val Asn Met Asp His Ser Glu Asn Ala 325 330 335 Ile Gly ValAla Ala Thr Met Ala His Glu Met Gly His Asn Phe Gly 340 345 350 Met ThrHis Asp Ser Ala Asp Cys Cys Ser Ala Ser Ala Ala Asp Gly 355 360 365 GlyCys Ile Met Ala Ala Ala Thr Gly His Pro Phe Pro Lys Val Phe 370 375 380Asn Gly Cys Asn Arg Arg Glu Leu Asp Arg Tyr Leu Gln Ser Gly Gly 385 390395 400 Gly Met Cys Leu Ser Asn Met Pro Asp Thr Arg Met Leu Tyr Gly Gly405 410 415 Arg Arg Cys Gly Asn Gly Tyr Leu Glu Asp Gly Glu Glu Cys AspCys 420 425 430 Gly Glu Glu Glu Glu Cys Asn Asn Pro Cys Cys Asn Ala SerAsn Cys 435 440 445 Thr Leu Arg Pro Gly Ala Glu Cys Ala His Gly Ser CysCys His Gln 450 455 460 Cys Lys Leu Leu Ala Pro Gly Thr Leu Cys Arg GluGln Ala Arg Gln 465 470 475 480 Cys Asp Leu Pro Glu Phe Cys Thr Gly LysSer Pro His Cys Pro Thr 485 490 495 Asn Phe Tyr Gln Met Asp Gly Thr ProCys Glu Gly Gly Gln Ala Tyr 500 505 510 Cys Tyr Asn Gly Met Cys Leu ThrTyr Gln Glu Gln Cys Gln Gln Leu 515 520 525 Trp Gly Pro Gly Ala Arg ProAla Pro Asp Leu Cys Phe Glu Lys Val 530 535 540 Asn Val Ala Gly Asp ThrPhe Gly Asn Cys Gly Lys Asp Met Asn Gly 545 550 555 560 Glu His Arg LysCys Asn Met Arg Asp Ala Lys Cys Gly Lys Ile Gln 565 570 575 Cys Gln SerSer Glu Ala Arg Pro Leu Glu Ser Asn Ala Val Pro Ile 580 585 590 Asp ThrThr Ile Ile Met Asn Gly Arg Gln Ile Gln Cys Arg Gly Thr 595 600 605 HisVal Tyr Arg Gly Pro Glu Glu Glu Gly Asp Met Leu Asp Pro Gly 610 615 620Leu Val Met Thr Gly Thr Lys Cys Gly Tyr Asn His Ile Cys Phe Glu 625 630635 640 Gly Gln Cys Arg Asn Thr Ser Phe Phe Glu Thr Glu Gly Cys Gly Lys645 650 655 Lys Cys Asn Gly His Gly Val Cys Asn Asn Asn Gln Asn Cys HisCys 660 665 670 Leu Pro Gly Trp Ala Pro Pro Phe Cys Asn Thr Pro Gly HisGly Gly 675 680 685 Ser Ile Asp Ser Gly Pro Met Pro Pro Glu Ser Val GlyPro Val Val 690 695 700 Ala Gly Val Leu Val Ala Ile Leu Val Leu Ala ValLeu Met Leu Met 705 710 715 720 Tyr Tyr Cys Cys Arg Gln Asn Asn Lys LeuGly Gln Leu Lys Pro Ser 725 730 735 Ala Leu Pro Ser Lys Leu Arg Gln GlnPhe Ser Cys Pro Phe Arg Val 740 745 750 Ser Gln Asn Ser Gly Thr Gly HisAla Asn Pro Thr Phe Lys Leu Gln 755 760 765 Thr Pro Gln Gly Lys Arg LysVal Ile Asn Thr Pro Glu Ile Leu Arg 770 775 780 Lys Pro Ser Gln Pro ProPro Arg Pro Pro Pro Asp Tyr Leu Arg Gly 785 790 795 800 Gly Ser Pro ProAla Pro Leu Pro Ala His Leu Ser Arg Ala Ala Arg 805 810 815 Asn Ser ProGly Pro Gly Ser Gln Ile Glu Arg Thr Glu Ser Ser Arg 820 825 830 Arg ProPro Pro Ser Arg Pro Ile Pro Pro Ala Pro Asn Cys Ile Val 835 840 845 SerGln Asp Phe Ser Arg Pro Arg Pro Pro Gln Lys Ala Leu Pro Ala 850 855 860Asn Pro Val Pro Gly Arg Arg Ser Leu Pro Arg Pro Gly Gly Ala Ser 865 870875 880 Pro Leu Arg Pro Pro Gly Ala Gly Pro Gln Gln Ser Arg Pro Leu Ala885 890 895 Ala Leu Ala Pro Lys Val Ser Pro Arg Glu Ala Leu Lys Val LysAla 900 905 910 Gly Thr Arg Gly Leu Gln Gly Gly Arg Cys Arg Val Glu LysThr Lys 915 920 925 Gln Phe Met Leu Leu Val Val Trp Thr Glu Leu Pro GluGln Lys Pro 930 935 940 Arg Ala Lys His Ser Cys Phe Leu Val Pro Ala 945950 955 9 3512 DNA homo sapiens 9 cacccgtccg ttctccatct ttcccgccttcctcaaccct cgggtgacct tccttcttca 60 tcctgcggag ccccggggct gagcggagcgtctcgacaga ggctggagcg ggtggggagg 120 cgcggggcga gccggggggt tccagacgcgcctccaccgc cgggcagtgg gcaggtatgg 180 ctgagggcgt gtgagcgccg agcgctaagggccgccgcca ccatgccagg gggcgcaggc 240 gccgcccggc tctgcttgct ggcgtttgccctgcagcccc tccggccgcg ggcggcgcgg 300 gagcctggat ggacaagagg aagtgaggaaggcagcccca agctgcagca tgaacttatc 360 atacctcagt ggaagacttc agaaagccccgtgagagaaa agcatccact caaagctgag 420 ctcagggtaa tggctgaggg gcgagaactgatcctggacc tggagaagaa tgagcaactt 480 tttgctcctt cctacacaga aacccattatacttcaagtg gtaaccctca aaccaccaca 540 cggaaattgg aggatcactg cttttaccacggcacggtga gggagacaga actgtccagc 600 gtcacgctca gcacttgccg aggaattagaggactgatta cggtgagcag caacctcagc 660 tacgtcatcg agcccctccc tgacagcaagggccaacacc ttatttacag atctgaacat 720 ctcaagccgc ccccgggaaa ctgtgggttcgagcactcca agcccaccac cagggactgg 780 gctcttcagt ttacacaaca gaccaagaagcgacctcgca ggatgaaaag ggaagattta 840 aactccatga agtatgtgga gctttacctcgtggctgatt atttagagtt tcagaagaat 900 cgacgagacc aggacgccac caaacacaagctcatagaga tcgccaacta tgttgataag 960 ttttaccgat ccttgaacat ccggattgctctcgtgggct tggaagtgtg gacccacggg 1020 aacatgtgtg aagtttcaga gaatccatattctaccctct ggtcctttct cagttggagg 1080 cgcaagctgc ttgcccagaa gtaccatgacaacgcccaat taatcacggg catgtccttc 1140 cacggcacca ccatcggcct ggcccccctcatggccatgt gctctgtgta ccagtctgga 1200 ggagtcaaca tggaccactc cgagaatgccattggcgtgg ctgccaccat ggcccacgag 1260 atgggccaca actttggcat gacccatgattctgcagatt gctgctcggc cagtgcggct 1320 gatggtgggt gcatcatggc agctgccactgggcacccct ttcccaaagt gttcaatgga 1380 tgcaacagga gggagctgga caggtatctgcagtcaggtg gtggaatgtg tctctccaac 1440 atgccagaca ccaggatgtt gtatggaggccggaggtgtg ggaacgggta tctggaagat 1500 ggggaagagt gtgactgtgg agaagaagaggaatgtaaca acccctgctg caatgcctct 1560 aattgtaccc tgaggccggg ggcggagtgtgctcacggct cctgctgcca ccagtgtaag 1620 ctgttggctc ctgggaccct gtgccgcgagcaggccaggc agtgtgacct cccggagttc 1680 tgtacgggca agtctcccca ctgccctaccaacttctacc agatggatgg taccccctgt 1740 gagggcggcc aggcctactg ctacaacggcatgtgcctca cctaccagga gcagtgccag 1800 cagctgtggg gacccggagc ccgacctgcccctgacctct gcttcgagaa ggtgaatgtg 1860 gcaggagaca cctttggaaa ctgtggaaaggacatgaatg gtgaacacag gaagtgcaac 1920 atgagagatg cgaagtgtgg gaagatccagtgtcagagct ctgaggcccg gcccctggag 1980 tccaacgcgg tgcccattga caccactatcatcatgaatg ggaggcagat ccagtgccgg 2040 ggcacccacg tctaccgagg tcctgaggaggagggtgaca tgctggaccc agggctggtg 2100 atgactggaa ccaagtgtgg ctacaaccatatttgctttg aggggcagtg caggaacacc 2160 tccttctttg aaactgaagg ctgtgggaagaagtgcaatg gccatggggt ctgtaacaac 2220 aaccagaact gccactgcct gccgggctgggccccgccct tctgcaacac accgggccac 2280 gggggcagta tcgacagtgg gcctatgccccctgagagtg tgggtcctgt ggtagctgga 2340 gtgttggtgg ccatcttggt gctggcggtcctcatgctga tgtactactg ctgcagacag 2400 aacaacaaac taggccaact caagccctcagctctccctt ccaagctgag gcaacagttc 2460 agttgtccct tcagggtttc tcagaacagcgggactggtc atgccaaccc aactttcaag 2520 ctgcagacgc cccagggcaa gcgaaaggtgttccttgact tgtgcgtaca ggtgatcaac 2580 actccggaaa tcctgcggaa gccctcccagcctcctcccc ggccccctcc agattatctg 2640 cgtggtgggt ccccacctgc accactgccagctcacctga gcagggctgc taggaactcc 2700 ccagggcccg ggtctcaaat agagaggacggagtcgtcca ggaggcctcc tccaagccgg 2760 ccaattcccc ccgcaccaaa ttgcatcgtttcccaggact tctccaggcc tcggccgccc 2820 cagaaggcac tcccggcaaa cccagtgccaggccgcagga gcctccccag gccaggaggt 2880 gcatccccac tgcggccccc tggtgctggccctcagcagt cccggcctct ggcagcactt 2940 gccccaaagg tgagtccacg ggaagccctcaaggtgaaag ctggtaccag agggctccag 3000 gggggcaggt gtagagttga gaaaacaaagcaattcatgc ttcttgtggt ctggactgaa 3060 cttccagaac aaaagccaag ggcaaaacattcatgtttct tggtgcccgc ttgactgtgg 3120 agttttggct tcatgtgaaa ggtgattcttagaatcctga gctgtggtgg cttcagtcct 3180 gcccctgcac ctgacctggg gagggaccctgagcaagtcc ctcttgagtc tgtttcctca 3240 tttgtacaga ggctatgatg agcagtagtaccagccccat caggttgttg tgaagagcaa 3300 ggaagtgcat tagtagaacc acctggcctgcgggaggctg tgtacacttg gcccctyctc 3360 gtacttcttc tgtgtggctg acagtctcagtttgccagtc ttggcartca ggtcagaaag 3420 ggatgaggtg aagaagctgg ctkrmcatgctgtattccca cgaggcattg agaaaactat 3480 ggactgtgct gctgccttar cagggaagggaa 3512

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence encoding an amino acid sequence drawn from the groupconsisting of SEQ ID NOS: 2, 4, 6, and
 8. 2. An isolated nucleic acidmolecule comprising a nucleotide sequence that: (a) encodes the aminoacid sequence shown in SEQ ID NO: 6; and (b) hybridizes under stringentconditions to the nucleotide sequence of SEQ ID NO: 5 or the complementthereof.
 3. An isolated nucleic acid molecule encoding SEQ ID NO:2. 4.An isolated nucleic acid molecule encoding SEQ ID NO:4.